Project Summary Translation of the genetic code from mRNA into protein ultimately determines the protein composition of the cell. Translation elongation and its fidelity are essential for human health, as mutations in translation factors can result in intellectual disability. Translation elongation is modulated by the choice of synonymous codons used to encode a polypeptide and is subject to multiple quality control mechanisms to prevent synthesis of aberrant proteins. In the yeast Saccharomyces cerevisiae, translation of CGA-CGA codon pairs is strongly inhibitory, much more so than any single codon. Inhibition is mediated by ribosomal protein Asc1 (human RACK1), which triggers engagement of the ribosome quality control (RQC) system when ribosomes collide. To define the scope and mechanisms of codon-mediated effects on translation, we recently used a high throughput assay of GFP variants to identify 17 strongly inhibitory codon pairs, 12 of which are among the most slowly translated codon pairs in yeast. We infer that these pairs are functionally important, as the most slowly translated pairs are highly conserved in the corresponding positions of genes in closely related species. We are also studying the crucial process of reading frame maintenance, one of the most basic functions of the ribosome. We had found that strains lacking Asc1 undergo extensive frameshifting at CGA codon repeats. Using a genetic selection, we recently identified two additional proteins that work together with Asc1 to prevent frameshifting at CGA repeats: uS3/Rps3, a universally conserved ribosomal protein, and Mbf1, an archaeal/eukaryotic conserved protein, whose role in translation is poorly understood. Despite intensive study of reading frame maintenance, this entire system involving Asc1, Mbf1, and Rps3, which is specific to eukaryotes, has never been studied. Additional preliminary results have implicated two other proteins in reading frame maintenance: eS26 and Gcn1. Ribosomal protein eS26 sits at the interface of collided ribosomes, which have recently been implicated in frameshifting. Gcn1 is a major regulator of a conserved stress response pathway, involved in sensing uncharged tRNA at the A-site of the ribosome when ribosomes are stalled due to amino acid starvation. Remarkably, three of the twelve most inhibitory codon pairs respond to the RQC system and require Mbf1 for reading frame maintenance, and nine other inhibitory codon pairs do not. The mechanisms by which these nine pairs exert their effects on translation are a mystery, but seem likely to involve central components of the translational control systems as many of these pairs are highly conserved and slowly translated. To follow up on these results we propose to 1. Determine the mechanisms by which Mbf1, Rps3 and Asc1 work to maintain the reading frame. 2. Investigate the roles of Rps26 and Gcn1 proteins in frameshifting. 3. Define the mechanisms by which distinct inhibitory codon pairs exert their effects.